Methods for testing effectiveness of a von Willebrand factor used in treating von Willebrand disease (VWD)

ABSTRACT

The invention generally relates to methods of testing the effectiveness of a von Willebrand factor (VWF) in treating von Willebrand disease (VWD) in a subject comprising measuring VWF cleavage fragments in a blood sample from the subject before and after treatment. In particular, the invention relates to methods of measuring VWF cleavage fragments, wherein an increase in VWF cleavage fragments after the treatment indicates that endogenous a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) in the subject is cleaving the VWF and wherein a decrease or absence of VWF cleavage fragments after the treatment indicates that endogenous ADAMTS13 in the subject is not cleaving the VWF.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/345,070, filed Nov. 7, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/549,404, filed on Nov. 20, 2014, now U.S. Pat.No. 9,488,651, which is a divisional of U.S. patent application Ser. No.14/078,324, filed on Nov. 12, 2013, now U.S. Pat. No. 9,110,08, which isa divisional of U.S. patent application Ser. No. 13/795,214, filed onMar. 12, 2013, now U.S. Pat. No. 8,623,612, which is a divisional ofU.S. patent application Ser. No. 12/630,509, filed on Dec. 3, 2009, nowU.S. Pat. No. 8,415,114, which claims benefit of U.S. ProvisionalApplication No. 61/120,202, filed on Dec. 5, 2008, each of which ishereby incorporated herein in its entirety by reference thereto.

FIELD OF THE INVENTION

The invention generally relates to methods of measuring cleaved vonWillebrand factor (VWF) fragments. More specifically, the inventionrelates to methods of measuring the ability of a disintegrin andmetalloproteinase with a thrombospondin type 1 motif, member 13(ADAMTS13) to cleave VWF in vivo. The invention also relates to the useof various animal models which demonstrate ADAMTS13 activity similar tothat of a human.

BACKGROUND OF THE INVENTION

Circulating von Willebrand factor (VWF) in healthy humans is composed ofa series of high molecular weight multimers ranging from about 450,000to about 20 million Dalton (Da) or even higher molecular weight uponrelease from storage pools. VWF mediates primary hemostasis supportingthe adhesion of platelets to damaged blood vessels. In addition to beingnecessary for platelet aggregation, VWF is required for thestabilization of circulating Factor VIII (FVIII). In von Willebranddisease (VWD), at least one of these functions of VWF is reduced,resulting in clinical symptoms of varying severity.

The degree of VWF multimerization plays an important role in primaryhemostatic function and correlates with the ability to promote plateletaggregation. The lack of high multimer forms of VWF results in decreasedplatelet aggregation, as seen in subjects with Type II VWD. On the otherhand, the accumulation of ultra-large VWF multimers can cause thrombosisin the microvasculature. In healthy individuals the multimeric size ofVWF is regulated by the presence of ADAMTS13. Due to ADAMTS13 cleavageof the VWF monomers between Tyr¹⁶⁰⁵ and Met¹⁶⁰⁶, the multimer pattern ofVWF shows a characteristic “triplet” structure. Individuals lackingADAMTS13 have an increased portion of ultra-large VWF multimers with areduced triplet structure. These individuals often develop a syndromecalled thrombotic thrombocytopenic purpura (TTP) that is characterizedby the formation of thrombi in the microvasculature with plateletconsumption.

ADAMTS13 can only cleave VWF when its conformation changes from aglobular to an extended form, a change which normally occurs only undershear stress. ADAMTS13 activity is usually measured in vitro underdenaturing conditions to induce the conformational change, or by using apeptide substrate.

Currently, no method is available to test the in vivo activity ofADAMTS13 in the presence of endogenous VWF. Thus, there exists a need inthe art to develop new methods of measuring the cleavage of VWF byADAMTS13. There also remains a need in the art to determine the efficacyof new recombinant VWF and ADAMTS13 products during preclinical andclinical studies. In addition, there is a need in the art for newmethods to test the effectiveness of new therapies in the treatment ofADAMTS13 deficiencies in vivo.

SUMMARY OF THE INVENTION

The invention addresses one or more needs in the art relating to methodsof measuring the in vivo activity of ADAMTS13 and methods of evaluatingnew types of recombinant von Willebrand Factor (VWF) and recombinantADAMTS13 in vivo for their subsequent administration to a subject inneed thereof

In one aspect, the invention includes methods of detecting VWF fragmentsin the blood of a subject. Such methods show the in vivo activity ofADAMTS13 by detection (i.e., visualization and even quantification) ofthe amount of circulating fragments of cleaved VWF. In one aspect, themethods are based on SDS-PAGE combined with immunoblotting usingspecific antibodies against VWF. In some aspects, the VWF antibodies arespecific for different fragments of VWF. Such antibodies are polyclonalor monoclonal. In further aspects, a blood sample of a subject isapplied to a gel, the gel is subjected to immunoblotting with a VWFantibody conjugated to a marker, and the marker is detected withenhanced chemiluminescence.

In another aspect, the invention includes methods for determiningaberrant ADAMTS13 activity in vivo comprising the step of measuring VWFcleavage fragments in a blood sample from a test subject, wherein achange in VWF cleavage fragment levels in the blood sample of the testsubject compared to VWF cleavage fragment levels in a blood sample froma control subject known to have normal ADAMTS13 activity indicatesaberrant in vivo ADAMTS13 activity in the test subject.

The invention also includes methods for measuring ADAMTS13 activity in ablood sample from a subject comprising the steps of: measuring VWFcleavage fragments in the blood sample; comparing the VWF cleavagefragments to a reference curve of completely degraded VWF; andquantifying the VWF cleavage fragments based on the reference curve,wherein an amount of VWF cleavage fragments correlates with an amount ofADAMTS13 activity.

In another aspect, the invention includes methods for testingeffectiveness of a treatment for increasing ADAMTS13 activity orconcentration in a subject comprising measuring VWF cleavage fragmentsin a blood sample from the subject before and after the treatment,wherein an increase in VWF cleavage fragments after the treatmentindicates that the treatment is effective in increasing ADAMTS13activity or concentration in the subject.

In an additional aspect, the invention includes methods for testingeffectiveness of a treatment for von Willebrand disease (VWD) associatedwith a deficiency or dysfunction of ADAMTS13 in a subject comprisingmeasuring VWF cleavage fragments in a blood sample from the subjectbefore and after treatment, wherein an increase in VWF cleavagefragments after the treatment indicates that the treatment is effectivein treating the disease.

In a further aspect, the invention includes methods for testingeffectiveness of a VWF used in treating VWD in a subject comprisingmeasuring VWF cleavage fragments in a blood sample from the subjectbefore and after treatment, wherein an increase in VWF cleavagefragments after the treatment indicates that endogenous ADAMTS13 in thesubject is cleaving the VWF and wherein a decrease or absence of VWFcleavage fragments after the treatment indicates that endogenousADAMTS13 in the subject is not cleaving the VWF.

In various aspects, the methods of the invention allow the comparison ofspecies-species interaction of various sources of VWF and ADAMTS13 fromthe different species or animal models.

In various aspects, the types of treatment included in the methods ofthe invention include the administration of ADAMTS13 to the subject.

The invention further includes methods for testing the effectiveness ofa treatment for thrombotic thrombocytopenic purpura (TTP) in a subjectcomprising measuring VWF cleavage fragments in a blood sample from thesubject before and after the treatment, wherein a decrease in the amountof ultra-large multimers of VWF with a reduced triplet structure afterthe treatment indicates that the treatment is effective in increasingADAMTS13 activity or concentration in the subject.

In various aspects of the invention, measuring of VWF cleavage fragmentsor VWF level comprises performing Western blot analysis with a VWFantibody to visualize VWF cleavage fragments. In one aspect, Westernblot analysis is carried out under non-reducing conditions to increasesensitivity. In other aspects, reducing conditions are also used. Infurther aspects of the methods of the invention, VWF fragments arevisualized through use of a VWF antibody conjugated to a marker. Invarious aspects, the marker is alkaline phosphatase (ALP) or horseradishperoxidase (HRP). In even further aspects, the marker is detected withenhanced chemiluminescence (ECL).

In some aspects of the invention, VWF multimers are visualized by usinghigh resolution horizontal SDS-agarose gel electrophoresis followed byimmunostaining with a polyclonal rabbit anti-human VWF antibody. Invarious other aspects of the invention, the VWF antibody is monoclonalor polyclonal. Other types of antibodies known in the art are alsocontemplated for use in the methods of the invention. In even furtheraspects, VWF cleavage fragment level is detected at a sensitivity levelof about 0.025 to about 0.05 Ag U/mL VWF.

In other aspects, the invention includes methods of assessing ADAMTS13activity in a subject comprising comparing total VWF and VWF cleavagefragment level in a blood sample of the subject to a reference curve ofincreasingly degraded or digested VWF, wherein the VWF cleavage fragmentlevel in the blood sample correlates to an ADAMTS13 activity deducedfrom the reference curve.

In various aspects, the VWF cleavage fragment level in the blood sampleof the test subject is increased compared to VWF cleavage fragment levelin a blood sample from a control subject. In other aspects, the VWFcleavage fragment level in the blood sample of the test subject isdecreased compared to VWF cleavage fragment level in a blood sample froma control subject.

In some aspects, a change in VWF cleavage fragment level is detected bymeasuring the level of one or more VWF fragments. In certain aspects,the VWF fragment that is measured is a 140 kDa VWF fragment or a 176 kDaVWF fragment. In particular aspects, the VWF fragment that is measuredis a 176 kDa VWF fragment.

The invention includes methods for measuring ADAMTS13 activity in asubject comprising the steps of: adding VWF to a blood sample from thesubject; measuring VWF cleavage fragments in the blood sample afterexposure of the sample in the presence and absence of shear stress;comparing the VWF cleavage fragments to a reference curve of completelydegraded VWF or to a reference curve from diluted human or animalplasma; and quantifying the VWF cleavage fragments based on thereference curve, wherein an amount of VWF cleavage fragments correlateswith an amount of ADAMTS13 activity in the sample. In one aspect, theVWF is an intact recombinant VWF (rVWF) that is not yet cleaved byADAMTS13. In some aspects, the shear stress comprises a shear rate ofabout 100 s-1 to about 10,000 s-1 at a temperature of about 20° C. toabout 40 ° C. for a period of time. In other aspects, the shear rate isabout 1,000 s-1 to about 8,000 s-1. In one aspect, the shear rate isabout 6,000 s-1. In various aspects, the temperature is about 30° C. toabout 40° C. In one aspect, the temperature is about 37° C. In someaspects, the period of time ranges from about 30 seconds to about 1hour. In other aspects, the time ranges from about 15 minutes to about30 minutes. In one aspect, the time is about 30 minutes. In anotheraspect, the time is about 15 minutes.

In various aspects, the blood sample in the methods of the invention isplasma or serum. In particular aspects, the blood sample is plasma. Inother aspects, the blood sample is serum. In other aspects, the bloodsample is plasma that also contains cellular components such asplatelets and white blood cells.

In further aspects, the subject in the methods of the invention is amammal. In some aspects the mammalian subject is human, rabbit, monkey,dog, rat, mouse, or pig. In other aspects, the mammalian subject ishuman, rabbit, monkey, or dog. In a particular aspect, the subject ishuman.

BRIEF DESCRIPTION OF THE DRAWING

A further illustration of the invention is given with reference to theaccompanying drawings, which are set out below in FIGS. 1-18.

FIGS. 1A-1B show the changes in multimeric structure of rVWF aftercleavage by human ADAMTS13.

FIGS. 2A-2B show specific cleavage of rVWF monomers by ADAMTS13 asdetected by immunoblotting with VWF antibodies.

FIG. 3 shows ADAMTS13-dependent cleavage of rVWF detected by residualVWF:CBA assay.

FIG. 4 shows ADAMTS13 activity, expressed as a percentage of normalhuman plasma (NHP), as measured by FRETS and VWF:CBA assays.

FIG. 5 illustrates VWF cleavage by ADAMTS13 using high resolutionmultimer analysis and demonstrates that rabbit plasma induceddegradation of rVWF and the formation of satellite bands similar tohuman plasma.

FIG. 6 shows the results of SDS-PAGE under non-reducing conditionsfollowed by immunoblotting with polyclonal anti-human VWF antibodylinked to HRP after rVWF was incubated with plasma from various animalspecies. The results indicated cleavage of human rVWF after incubationwith plasma from human, rabbit, monkey, pig, or dog, but not with guineapig, rat, or mouse.

FIG. 7 shows the results of SDS-PAGE under reducing conditions followedby immunoblotting with monoclonal VWF antibodies. The asterisk denotesreactions of the goat anti-mouse IgG antibody (secondary) withendogenous mouse plasma IgGs.

FIGS. 8A, 8B and 8C show specific in vivo cleavage of rVWF (1200 IUVWF:RCo/kg) by ADAMTS13 (140 kDa and 176 kDa fragments) in rabbits. VWFfragments were detected with monoclonal antibodies by immunoblottingwith reducing SDS-PAGE (FIGS. 8A and B). As controls, uncleaved rVWF andrVWF cleaved with rADAMTS13 in vitro are shown. Characteristic changesin multimer pattern shortly after rVWF administration are also seen(FIG. 8C).

FIGS. 9A, 9B and 9C show specific in vivo cleavage of rVWF (2000 IUVWF:RCo/kg) by ADAMTS13 in VWF-deficient and ADAMTS13-deficient mice.The 176 kDa VWF cleavage fragment was not visible in either mouse strainusing the monoclonal antibody specific to the C-terminal fragment (FIG.9A). The asterisk denotes reactions of the goat anti-mouse IgG antibodywith mouse plasma IgGs. No detectable changes in rVWF multimer patternwere observed (FIG. 9B). 140 kDa and 176 kDa homodimers were onlydetectable in VWF-deficient, but not in ADAMTS13-deficient mice usingthe more sensitive (but less specific) polyclonal antibody undernon-reducing conditions (FIG. 9C).

FIG. 10 shows a direct comparison of the efficiency of ADAMTS13 cleavagein various animal models using non-reducing SDS-PAGE after equal amountsof VWF:Ag were loaded. In rabbit plasma samples, a stronger bandcorresponding to the 176 kDa VWF cleavage product was detectablecompared to the VWF-deficient mouse sample. No cleavage was detectablein ADAMTS13-deficient mouse plasma. Co-injection of rVWF with humanrADAMTS13 induced cleavage of rVWF.

FIGS. 11A-11B show results of Western blot detection of VWF cleavagefragments in Cynomolgus plasma after a single dose injection of rVWF(100 IU/kg). Completely and partially ADAMTS13-degraded rVWF fragments(1 Ag U/mL) were measured in Cynomolgus plasma after a single doseinjection of rVWF after 15 seconds of exposure (FIG. 11A) with therabbit anti-human VWF antibody. The intensity of a 176 kDa dimer bandincreased after injection of rVWF. The 176 kDa VWF dimer was greaterthan baseline (pre-injection) even 24 h after VWF injection (FIG. 11A),when no elevated VWF antigen (VWF:Ag) was measured (FIG. 11B).

FIGS. 12A-12B show completely and partially ADAMTS13-degraded rVWF (1 AgU/mL) measured in buffer and in VWF-deficient plasma with Westernblotting under non-reducing conditions after 15 seconds of exposure withthe polyclonal rabbit anti-human VWF antibody. Reducing conditions werenot sensitive enough to visualize results. No differences betweendilutions in buffer (FIG. 12A) and in VWF-deficient plasma (FIG. 12B)were detected.

FIGS. 13A-13B show that VWF cleavage fragments were detected in plasmaby increasing the sensitivity of the assay by increasing exposure time.Completely and partially ADAMTS13-degraded rVWF (1 Ag U/mL) weremeasured in buffer and VWF-deficient plasma with Western blotting undernon-reducing conditions after 15 seconds (FIG. 13A) and 60 seconds (FIG.13B) of exposure with the rabbit anti-human VWF antibody.

FIGS. 14A-14B show results of optimizing sensitivity of Western blotanalysis by carrying out sample dilutions and increasing blot exposuretimes. Normal human plasma was diluted 20-fold and a 176 kDa cleavageproduct was well detected after a two minute exposure time in 20-40 nLnormal human plasma (FIG. 14A). No ADAMTS13-specific band (VWF cleavageproduct) could be seen in VWF-deficient plasma (FIG. 14A). Fullydegraded rVWF (1 Ag U/mL) was diluted 40-fold (0.5 nL to 20 nL) andcould be detected at a level of sensitivity of about 0.0006 U/mL (0.5nL/0.8 μL plasma) (FIG. 14B).

FIGS. 15A-15B show the quantification of VWF cleavage by ADAMTS13 inplasma. A 176 kDa VWF cleavage product was well detected in normal humanplasma (about 0.025 Ag U/mL VWF) (FIG. 15A). Approximately 1-2% ofC-terminal dimers (the 176 kDa ADAMTS13-specific cleavage product) werefound in human normal plasma when calculated from a reference curve(FIG. 15B) constructed from the band intensity of different amounts ofcompletely degraded rVWF (1 Ag U/mL).

FIGS. 16A-16B show the results of Western blot detection of theC-terminal VWF cleavage fragment in human plasma after administration of7.5 (FIG. 16A) and 20 (FIG. 16B) IU VWF:RCo/kg to subjects in a clinicalphase I trial. The ADAMTS13-dependent rVWF fragment was detectable inplasma with the rabbit anti-human VWF antibody already 15 minutespost-treatment. The intensity of the 176 kDa dimer band remained abovebackground for approximately 1 hour (7.5 IU rVWF) and 32 hours (20 IUrVWF).

FIG. 17 shows the results of Western blot detection of the C-terminalVWF cleavage fragment after in vitro cleavage of 1 IU/mL rVWF by 0.2U/mL of human recombinant ADAMTS13, human plasma-derived ADAMTS13, andnormal human plasma in the presence and absence of shear stress.

FIGS. 18A-18B show the changes in multimeric structure of rVWF at low(FIG. 18A) and high (FIG. 18B) resolution after in vitro cleavage byhuman ADAMTS13 in the presence and absence of shear stress.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of measuring ADAMTS13 activity bymeasuring the in vivo cleavage of VWF or rVWF by ADAMTS13. The inventionalso provides methods for determining if VWF is normally processed. Theinvention addresses a need in the art for improved methods to testeffectiveness of new therapies in treatment of von Willebrand disease(VWD) and in treatment of thrombotic thrombocytopenic purpura (TTP) andother disorders associated with aberrant levels of ADAMTS13.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The following referencesprovide one of skill with a general definition of many of the terms usedin this invention: Singleton, et al., DICTIONARY OF MICROBIOLOGY ANDMOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE ANDTECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R.Rieger, et al. (eds.), Springer Verlag (1991); and Hale and Marham, THEHARPER COLLINS DICTIONARY OF BIOLOGY (1991).

It is noted here that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

It also is specifically understood that any numerical value recitedherein includes all values from the lower value to the upper value,i.e., all possible combinations of numerical values between the lowestvalue and the highest value enumerated are to be considered to beexpressly stated in this application. For example, if a concentrationrange is stated as about 1% to 50%, it is intended that values such as2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated inthis specification. The values listed above are only examples of what isspecifically intended.

Ranges may be expressed herein as from “about” or “approximately” oneparticular value and/or to “about” or “approximately” another particularvalue. When such a range is expressed, another embodiment includes fromthe one particular value and/or to the other particular value.Similarly, when values are expressed as approximations, by use of theantecedent “about,” it will be understood that the particular valueforms another embodiment.

The term “aberrant” refers to abnormal, atypical, or unnatural level oractivity of a polypeptide, protein or enzyme in a test subject comparedto the level or activity of the polypeptide, protein or enzyme in anormal or control subject. Such abnormal level or activity may reflect alower level, a lower activity, or a complete deficiency.

The term “VWF cleavage fragment” or “VWF fragments” or “VWF cleavageproducts” are used interchangeably herein and refer to fragments of VWFwhich result from protease cleavage. In one aspect, the proteasecleaving VWF is ADAMTS13. ADAMTS13, also called VWF-cleaving protease(VWFCP), is a zinc-containing metalloprotease enzyme that cleaves VWF.ADAMTS13 is secreted in blood and degrades large VWF multimers,decreasing their activity. ADAMTS13 consists of multiple structural andfunctional domains, and these domains may participate in the recognitionand binding of ADAMTS13 to VWF. The term “multimers” or “multimer forms”are used interchangeably herein. The ULVWF multimers are cleaved byADAMTS13 as they are secreted from endothelial cells. Thus, the terms“ADAMTS13” and “VWFCP” are used interchangeably.

The term “visualized” or “detected” are used interchangeably herein whendiscussing the examination of VWF cleavage fragment level(s) onimmunoblots or Western blots. Likewise, the term “level” or “levels”refers to the amount or concentration of VWF visualized, detected, ormeasured in a blot or assay.

The term “subject” or “test subject” or “subject in need thereof” may beused interchangeably herein and refers to any mammal. In variousaspects, the subject is human, rabbit, monkey, or dog. Like humans, dogsare also known to suffer from VWD.

The term “blood” or “blood sample” may be used interchangeably herein.In various aspects, the blood is plasma or serum. Thus, the terms“blood”, “blood sample”, “plasma”, “plasma sample”, “serum” and “serumsample” are used interchangeably herein.

The term “endogenous” refers to a polypeptide or polynucleotide or othercompound that is expressed naturally in a host organism, or originateswithin a cell, tissue or organ. “Exogenous” refers to a polypeptide,polynucleotide or other compound that originates from outside of a cell,tissue or organ outside of a host organism.

The term “polypeptide” refers to a polymer composed of amino acidresidue linked via peptide bonds. Synthetic polypeptides aresynthesized, for example, all or in part using an automated polypeptidesynthesizer. The term “protein” typically refers to large polypeptides.The term “peptide” typically refers to short polypeptides.

The term “ADAMTS13” or “recombinant ADAMTS13” or “rADAMTS13” may be usedinterchangeably herein and refers to a disintegrin and metalloproteinasewith a thrombospondin type 1 motif, member 13 polypeptide.

The term “VWF” or “recombinant VWF” or “rVWF” may be usedinterchangeably herein and refers to von Willebrand factor polypeptide.

The term “agent” or “compound” describes any molecule with thecapability of affecting a biological parameter in the mammalian subjectof the invention.

The methods of the invention included the use of various assays tomeasure ADAMTS13 activity. In some aspects, cleavage of rVWF by ADAMTS13is carried out as described by Gerritsen et al. (Thromb. Haemost. 82:1386-1389, 1999) with minor modifications. Recombinant VWF is digestedwith Ba²⁺-activated ADAMTS13 under denaturing conditions. Plasma samplesare diluted 1:20 in 5 mM Tris, 1.5 M urea, pH 8.0 (50 mU/mL ADAMTS13activity), mixed with 9.3 mM BaCl₂ to activate ADAMTS13 and 1 IU/mL ofhuman rVWF (final concentration) and incubated at 37° C. for 24 h. Thereactions are stopped by adding Na₂SO₄ (8.25 mM). The solution iscentrifuged and the resulting supernatant used for further analysis. Asa positive control, 1 IU/mL of rVWF is incubated with human rADAMTS13(50 mU/mL) under conditions that are otherwise identical to those usedfor the test item.

In other aspects of the invention, VWF antigen is determined in plasmasamples. VWF:Ag was determined with an ELISA using commerciallyavailable polyclonal rabbit anti-VWF antibodies (Dako, Glostrup,Denmark) as described by Tan et al. (Thromb. Res. 121: 519-526, 2008).

In another aspect of the invention, ADAMTS13 activity is measured usinga fluorescent-labeled synthetic VWF peptide composed of 73 amino acids(FRETS-VWF73, Peptides Institute, Osaka, Japan) according tomanufacturer's instructions. Plasma samples are measured against areference curve of diluted normal human plasma.

In even further aspects of the invention, determination ofcollagen-binding activity (VWF:CBA) is carried out. Binding of rVWF tocollagen in some aspects is determined according to a publishedELISA-based method (Turecek et al., Semin. Thromb. Hemost. 28: 149-160,2002). In some aspects, the collagen source is human collagen type IIIfrom Southern Biotechnology Associates (Birmingham, Ala.). Bound rVWF isdetected with polyclonal anti-VWF antibody conjugated with HRP (Dako).

In yet another aspect of the invention, VWF multimer analysis is carriedout. In some aspects, the multimeric structure of rVWF is analyzed byhigh-density horizontal SDS agarose gel electrophoresis as described byTurecek et al. (Blood 90: 3555-3567, 1997). Samples are diluted to 1.0IU/mL VWF:Ag, and incubated with Tris-EDTA-SDS buffer. The multimerscontained in the solution are separated under non-reducing conditions ona 2.5% high-resolution agarose gel. VWF multimers are visualized in thegel by immunostaining with a polyclonal rabbit anti-human VWF antibody(Dako), followed by alkaline-phosphatase (ALP)-conjugated anti-rabbitIgG using the ALP color development kit from Bio-Rad (Richmond, Calif,USA).

The invention also includes the analysis of specific ADAMTS13 cleavageproducts. ADAMTS13-mediated cleavage in some aspects is detected bySDS-PAGE and Western blot analysis using either a polyclonal anti-humanVWF antibody (Dako) or in some aspects one of the following mousemonoclonal antibodies: VW33-5, directed against the V8 protease fragmentI of VWF (TaKaRa Bio Europe, Saint-Germain-en-Laye, France); VW92-3,directed against the V8 protease fragment III of VWF under non-reducingconditions (TaKaRa Bio Europe); EsvWF10, which recognizes the Al domainof VWF under reducing conditions (American Diagnostica, Stamford,Calif.), and N10, which detects the epitope generated upon ADAMTS13cleavage in the N-terminal fragment (Kato et al., Transfusion 46:1444-1452, 2006). In some aspects, antibodies are used withALP-conjugated goat anti-mouse secondary antibodies and the ALPdetection kit (Bio-Rad). In other aspects, a horseradish peroxidase(HRP)-conjugated polyclonal anti-VWF antibody from rabbit (Dako) is usedwith the ECL detection system (GE Healthcare, Munich, Germany) tocircumvent secondary antibodies reacting with endogenous immunoglobulinsin mouse and rabbit plasma.

The invention also includes methods of carrying out in vivo cleavage ofrVWF in different animal species. In some aspects, in vivosusceptibility of human rVWF to ADAMTS13 cleavage is determined byinjecting rabbits and cynomolgus monkeys with rVWF at doses of 1200,600, 300, and 100 VWF:RCo II/kg body weight (BW) and VWF- orADAMTS13-deficient mice with rVWF at a dose of 2000 VWF:RCo IU/kg BW.Blood samples are taken at various time points. Mouse samples areimmuno-depleted with protein G sepharose (Invitrogen, Carlsbad, Calif.)prior to analysis in some experiments.

The methods of the invention include the diagnosis and testingeffectiveness of a treatment for a disease or disorder associated withaberrant ADAMTS13 activity. Such diseases or disorders includethrombotic thrombocytopenic purpura (TTP or Moschcowitz disease). TTP isa rare disorder of the blood-coagulation system, causing extensivemicroscopic blood clots to form in the small blood vessels throughoutthe body. Most cases of TTP arise from deficiency or inhibition of theenzyme ADAMTS13, which is responsible for cleaving large multimers ofVWF. Red blood cells passing the microscopic clots are subjected toshear stress which leads to hemolysis. Reduced blood flow and cellularinjury results in end organ damage. Current therapy is based on supportand plasmapheresis to reduce circulating antibodies against ADAMTS13 andreplenish blood levels of the enzyme.

It has been found that subjects with congenital TTP or acquired TTP areseverely deficient in ADAMTS13. ADAMTS13 is a metalloproteinaseresponsible for the breakdown of VWF, a protein that links platelets,blood clots, and the blood vessel wall in the process of bloodcoagulation. Very large VWF molecules are more prone to lead tocoagulation. Hence, without proper cleavage of VWF by ADAMTS13,coagulation occurs at a higher rate, especially in the part of the bloodvessel system where VWF is most active due to high shear stress: in themicrovasculature. Congenital ADAMTS13 deficiency is caused by mutationsof the ADAMTS13 gene. Subjects with the familial form have severeprotease deficiency. ADAMTS13 gene mutation in familial TTP causesinactivity or decreased activity of ADAMTS13. Acquired deficiency occurswith the production of autoantibodies inhibiting ADAMTS13 activity.Acquired TTP is idiopathic and secondary to complications such asautoimmune disease, malignancy, stem cell transplantation, pregnancy(especially the third trimester), certain drugs (including ticlopidine,mitomycin, clopidogrel, and cyclosporine) or infection. The inventionprovides methods of measuring ADAMTS13 activity in blood and for testingeffectiveness of treatment for diseases associated with abnormalADAMTS13 levels or activity in the blood.

Deficiency of ADAMTS13 was originally discovered in Upshaw-Shulmansyndrome, the recurring familial form of thrombotic thrombocytopenicpurpura (TTP). By that time it was already suspected that TTP occurredin the autoimmune form as well, owing to its response to plasmapheresisand characterization of IgG inhibitors. Since the discovery of ADAMTS13,specific epitopes on its surface have been shown to be the target ofinhibitory antibodies.

More than 70 mutations in the ADAMTS13 gene have been reported in peoplewith the familial form of TTP. Most of these mutations change singleamino acids in the ADAMTS13 enzyme. Other mutations lead to theproduction of an abnormally small version of ADAMTS13 that cannotfunction properly. Mutations in the ADAMTS13 gene severely reduce theactivity of the ADAMTS13 enzyme. As a result, VWF is not processednormally in the bloodstream. If VWF is not processed normally byADAMTS13, it promotes the formation of abnormal clots throughout thebody by inducing platelets to stick together and adhere to the walls ofblood vessels, even in the absence of injury. Additional factors such aspregnancy, diarrhea, surgery, and infection likely play a role intriggering abnormal clotting. Blood clots can block blood flow throughsmall vessels, causing damage to the brain, kidneys, heart, and otherorgans. Abnormal clotting also causes other complications associatedwith TTP.

The TTP syndrome is characterized by microangiopathic hemolysis andplatelet aggregation/hyaline thrombi whose formation is unrelated tocoagulation system activity. Platelet microthrombi predominate; theyform in the microcirculation (i.e., arterioles, capillaries) throughoutthe body causing partial occlusion of vessels. Organ ischemia,thrombocytopenia, and erythrocyte fragmentation (i.e., schistocytes)occur. The thrombi partially occlude the vascular lumina with overlyingproliferative endothelial cells. The endothelia of the kidneys, brain,heart, pancreas, spleen, and adrenal glands are particularly vulnerableto TTP. The liver, lungs, gastrointestinal tract, gallbladder, skeletalmuscles, retina, pituitary gland, ovaries, uterus, and testes are alsoaffected to a lesser extent. No inflammatory changes occur.

In 1982, Moake and his colleagues observed ultra-large VWF (ULVWF)multimers in the plasma of four subjects with relapsing TTP (Moake J L,Semin. Hematol. 34:83-89, 1997; Moake J L, Semin. Hematol. 41:4-14,2004). These multimers were the same size as those noted in theendothelial cells. The plasma of normal individuals has much smallerVWF. Moake suggested that there was a deficiency in an enzyme thatreduces the large VWF to its normal size in plasma in subjects with TTP.Also noted was that this large VWF has a greater ability to adhere withplatelets mediating a thrombus formation.

The present section provides a description of the relationship betweenthe biological function of ADAMTS13 and the existence of ULVWF multimersand the occurrence of TTP or TTP-like clinical symptoms to the extentthat such a description will facilitate a better understanding of themethods of the invention. There is a relationship between the biologicalfunction of ADAMTS13 and the existence of ULVWF multimers and theoccurrence of TTP or TTP-like clinical symptoms. The pathogenesis of TTPis due to the platelet clumping in the microvasculature. There is anincreased adherence of the ULVWF multimers leading to the formation ofplatelet thrombi due to the lack of a functioning proteolytic enzyme(ADAMTS13) to cleave these multimers.

TTP also may be related to cancer, chemotherapy, HIV infection, hormonereplacement therapy and estrogens, and a number of commonly usedmedications (including ticlopidine, clopidogrel, and cyclosporine A).Systemic connective tissue diseases are other conditions besides TTPthat are associated in some instances with low but detectable levels ofADAMTS13. Recombinant ADAMTS13 (rADAMTS13) is one of many therapiesbeing tested in the treatment of TTP. The invention includes methods oftesting the effectiveness of rADAMTS13 and all therapies associated withADAMTS13-associated diseases.

A low level of ADAMTS13 causes clotting substances (platelets) in theblood to clump. As the platelets clump together, there are fewerplatelets available in the bloodstream. This clumping, or aggregation,can lead to bleeding under the skin and purple-colored spots calledpurpura. It also can cause red blood cells to break apart (undergohemolysis) as they are subjected to shear stress as they pass themicroscopic platelet clots. Red blood cells are thus destroyedprematurely. Reduced blood flow and cellular injury results in end organdamage.

Levels of human ADAMTS13 antigen may be determined by ELISA (Rieger etal., Thromb. Haemost. 95:212-220, 2006). ADAMTS13 activity may bemeasured based on decreased collagen binding affinity of degraded VWF(Gerritsen et al., Thromb. Haemost. 82:1386-1389, 1999), which is afunctional assay based on the preferential binding ofhigh-molecular-weight forms of VWF to collagen. In this assay, thediluted plasma sample to be tested is added to normal plasma in whichprotease activity had been abolished. The VWF present in theprotease-depleted plasma is digested by the VWF-cleaving protease in thetest plasma. The proteolytic degradation leads to low-molecular-weightforms of VWF, which show impaired binding to microtiter plates coatedwith human collagen type III. The collagen-bound VWF is quantified usinga peroxidase-conjugated rabbit antibody against human VWF. The values ofVWF-cleaving protease activity in tested plasma samples are read from acalibration curve achieved by incubating the VWF-substrate withdilutions of a normal human plasma pool (NHP).

Current purpura or TTP therapy is based on support and plasmapheresis toreduce circulating antibodies against ADAMTS13 and replenish bloodlevels of ADAMTS13. Plasma exchange has been the first-line therapy forTTP since 1991. Congenital deficiency can replace the deficiency andmutations in the ADAMTS13 gene by plasma infusion. Acquired deficiencycan remove the inhibitor of ADAMTS13 by plasmapheresis. However, plasmaexchange is more effective treatment than plasma infusion. Thislife-threatening condition may have a positive outcome if recognizedearly and medical intervention is initiated early.

In addition, an increase in circulating levels of VWF and a decrease inADAMTS13 activity in humans are considered risk factors for ischemicstroke (Zhao et al., American Society of Hematology, Abstract 259, Dec.6-9, 2008, San Francisco, Calif.). Thus, the methods of measuringADAMTS13 activity in vivo herein also have application in strokediagnosis and therapy.

The invention also includes methods for diagnosing abnormal processingof VWF or deficiencies in ADAMTS13 concentration or activity, andtesting therapies to increase ADAMTS13 concentration or activity used inthe development of new therapies in the treatment of TTP and otherADAMTS13-related pathologies or disorders. In various aspects, otherADAMTS13-related pathologies or disorders include diseases which arecharacterized by abnormal levels of VWF or abnormal processing of VWF.

The present section provides a description of VWF syndrome to the extentthat such a description will facilitate a better understanding of themethods of the invention. VWF syndrome manifests clinically when thereis either an underproduction or an overproduction of VWF. Overproductionof VWF causes increased thrombosis (formation of a clot or thrombusinside a blood vessel, obstructing the flow of blood) while reducedlevels of, or lack of, high-molecular weight forms of VWF causesincreased bleeding and an increased bleeding time due to inhibition ofplatelet aggregation and wound closure. The methods of the invention canbe used in the diagnosis and treatment of various types of VWF syndrome.

A VWF deficiency may also cause a phenotypic Hemophilia A since VWF isan essential component of functional FVIII. In these instances, thehalf-life of Factor VIII is reduced to such an extent that its functionin the blood coagulation cascade is impaired. Subjects suffering fromVWD or VWF syndrome frequently exhibit an FVIII deficiency. In thesesubjects, reduced FVIII activity is not the consequence of a defect ofthe X chromosomal gene, but an indirect consequence of quantitative andqualitative change(s) of VWF in plasma. The differentiation betweenHemophilia A and VWD may normally be effected by measuring the VWFantigen or by determining the ristocetin-cofactor activity. Ristocetincofactor activity is measured by adding ristocetin and a plateletsubstrate to the subject's plasma. Ristocetin enhances binding of VWF tothe platelet glycoprotein Ib receptor, resulting in agglutination. Thesubject's VWF will support the platelet agglutination induced by theristocetin as measured by a change in light transmission. Therefore,this assay is an in vitro measurement of the functional activity of thesubject's VWF. Both the VWF antigen content and the ristocetin cofactoractivity are lowered in most VWD subjects, whereas they are normal inHemophilia A subjects.

VWD is an inherited bleeding disorder that is caused by deficiency ordysfunction of VWF. Therefore, defects in VWF can cause bleeding byimpairing platelet adhesion or by reducing the concentration of FVIII.VWD is diagnosed after a clinical and physical review, with personal andfamilial evidence of (primarily mucocutaneous) bleeding, and confirmedby laboratory testing. Laboratory testing typically entails initialplasma testing of factor VIII coagulant (FVIII:C), von Willebrand factor(VWF) protein (antigen; VWF:Ag), and VWF function or activity, which isassessed using the ristocetin cofactor (VWF:RCo) assay or the collagenbinding assay (VWF:CBA). The VWF:CBA is based on measurement of thequantity of VWF bound to collagen, similar to the procedure for anenzyme-linked immunosorbent assay. Additional laboratory testing cancomprise a battery of confirmatory and VWD subtype assisting assays,including assessment of VWF:multimers.

More specifically, the VWF:Ag assay is a quantitative assay and providesa measure of the overall level of VWF present in a patient's plasma; itis not a functional assay and yields no information concerning thequality of the VWF present. The VWF:CBA assay is a functional assaywhich provides information on the quality of VWF present. The VWF:Ag andVWF:CBA are complementary assays and, in various aspects, are used incombination. The VWF:RCo assay is both a quantitative and qualitativeassay that provides information about the presence of VWF that liesbetween that provided individually by the VWF:Ag and VWF:CBA assays. TheVWF:multimer assay is a qualitative procedure, and is semi-quantitative.The VWF:multimer assay provides a snap-shot of the VWF present. Theabove-described assays are well known in the art for testing VWF invitro.

Methods provided also include, in various aspects, use of VWF. All formsof VWF and recombinant VWF are contemplated for use in the methods ofthe invention. In some aspects, VWF used in the methods of the inventioninclude, but are not limited to: HUMATE-P®; and, IMMUNATE®, INNOBRAND®,and 8Y®.

In various aspects of the methods of the invention, recombinant VWF isadministered to a subject. Recombinant VWF is administered at a dose ofat least about 10 RCoU/kg BW, of at least about 20 RCoU/kg BW, of atleast about 30 RCoU/kg BW, of at least about 40 RCoU/kg BW, of at leastabout 50 RCoU/kg BW, of at least about 60 RCoU/kg BW, of at least about70 RCoU/kg BW, of at least about 80 RCoU/kg BW, of at least about 90RCoU/kg BW, of at least about 100 RCoU/kg BW, of at least about 150RCoU/kg BW, of at least about 200 RCoU/kg BW, of at least about 250RCoU/kg BW, of at least about 300 RCoU/kg BW, of at least about 350RCoU/kg BW, of at least about 400 RCoU/kg BW, of at least about 450RCoU/kg BW, of at least about 500 RCoU/kg BW, of at least about 550RCoU/kg BW, of at least about 600 RCoU/kg BW, of at least about 650RCoU/kg BW, of at least about 700 RCoU/kg BW, of at least about 750RCoU/kg BW, of at least about 800 RCoU/kg BW, of at least about 850RCoU/kg BW, of at least about 900 RCoU/kg BW, of at least about 950RCoU/kg BW, of at least about 1000 RCoU/kg BW, of at least about 1200RCoU/kg BW, of at least about 1400 RCoU/kg BW, of at least about 1600RCoU/kg BW, of at least about 1800 RCoU/kg BW, of at least about 2000RCoU/kg BW, of at least about 2500 RCoU/kg BW, of at least about 3000RCoU/kg BW, of at least about 3500 RCoU/kg BW, of at least about 4000RCoU/kg BW, of at least about 4500 RCoU/kg BW, of at least about 5000RCoU/kg BW, of at least about 6000 RCoU/kg BW, of at least about 7000RCoU/kg BW, of at least about 8000 RCoU/kg BW, of at least about 9000RCoU/kg BW, of at least about 10000 RCoU/kg BW, of at least about 20000RCoU/kg BW, of at least about 50000 RCoU/kg BW, and of at least about100000 RCoU/kg BW, and up to more than 100000 RCoU/kg BW.

In various other aspects of the methods of the invention, recombinantADAMTS13 is administered to a subject. Recombinant human ADAMTS13 hasbeen described (Plaimauer et al., Blood 100:3626-3632, 2002).Recombinant human ADAMTS13 is not yet available commercially for humanadministration, but the invention includes the use of such rADAMTS13 inclinical trials and when it is commercially available. In subjects withan inherited ADAMTS13 deficiency, normal human plasma is used as asource of ADAMTS13 and contains IU/mL of ADAMTS13. Purifiedplasma-derived or recombinant ADAMTS13 is currently available for use inanimals at a dose range of 100-500 U/kg BW. The methods of the inventioncontemplate the use of any of these sources administered at anappropriate dose of IU/mL or U/kg BW. In one aspect, the inventionincludes the administration or rADAMTS13 at a dose of at least about 10U/kg BW, of at least about 20 U/kg BW, of at least about 30 U/kg BW, ofat least about 40 U/kg BW, of at least about 50 U/kg BW, of at leastabout 60 U/kg BW, of at least about 70 U/kg BW, of at least about 80U/kg BW, of at least about 90 U/kg BW, of at least about 100 U/kg BW, ofat least about 150 U/kg BW, of at least about 200 U/kg BW, of at leastabout 250 U/kg BW, of at least about 300 U/kg BW, of at least about 350U/kg BW, of at least about 400 U/kg BW, of at least about 450 U/kg BW,of at least about 500 U/kg BW, of at least about 550 U/kg BW, of atleast about 600 U/kg BW, of at least about 650 U/kg BW, of at leastabout 700 U/kg BW, of at least about 750 U/kg BW, of at least about 800U/kg BW, of at least about 850 U/kg BW, of at least about 900 U/kg BW,of at least about 950 U/kg BW, of at least about 1000 U/kg BW, of atleast about 1200 U/kg BW, of at least about 1400 U/kg BW, of at leastabout 1600 U/kg BW, of at least about 1800 U/kg BW, of at least about2000 U/kg BW, of at least about 2500 U/kg BW, of at least about 3000U/kg BW, of at least about 3500 U/kg BW, of at least about 4000 U/kg BW,of at least about 4500 U/kg BW, of at least about 5000 U/kg BW, of atleast about 6000 U/kg BW, of at least about 7000 U/kg BW, of at leastabout 8000 U/kg BW, of at least about 9000 U/kg BW, of at least about10000 U/kg BW, of at least about 20000 U/kg BW, of at least about 50000U/kg BW, and of at least about 100000 U/kg BW, and up to more than100000 U/kg BW.

In various aspects of the methods of the invention, the methods arecarried out at a range of temperatures. In certain aspects, theinvention includes methods comprising temperatures of about 20° C.,about 21° C., about 22° C., about 23° C., about 24° C., about 25° C.,about 26° C., about 27° C., about 28° C., about 29° C., about 30° C.,about 31° C., about 32° C., about 33° C., about 34° C., about 35° C.,about 36° C., about 37° C., about 38° C., about 39° C., about 40° C.,about 41° C., about 42° C., about 43° C., about 44° C., about 45° C.,about 46° C., about 47° C., about 48° C., about 49° C., about 50° C.,about 51° C., about 52° C., about 53° C., about 54° C., about 55° C.,about 56° C., about 57° C., about 58° C., about 59° C., about 60° C.,about 61° C., about 62° C., about 63° C., about 64° C., about 65° C.,about 66° C., about 67° C., about 68° C., about 69° C., about 70° C.,about 71° C., about 72° C., about 73° C., about 74° C., about 75° C.,about 76° C., about 77° C., about 78° C., about 79° C., about 80° C.,about 90° C., and about 100° C. In one aspect, the invention includesmethods comprising temperatures of about 20° C. to about 40° C. In aparticular aspect, the invention includes methods comprisingtemperatures of about 30° C. to about 35° C. In one aspect, theinvention includes methods comprising a temperature of about 32° C.

In some aspects of the invention, the methods provided are carried outover a period of time. In various aspects, the invention includesmethods comprising times of about 5 seconds, about 10 seconds, about 15seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 35seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours,about 11 hours, about 12 hours, about 24 hours, about 36 hours, about 48hours, about 72 hours, and about 96 hours. In certain aspects, the timeranges from about 10 seconds to about 3 hours. In particular aspects,the time ranges from about 30 seconds to about 1 hour. In moreparticular aspects the time ranges from about 15 minutes to about 30minutes. In one aspect, the time is about 30 minutes. In another aspect,the time is about 15 minutes.

In some aspects, the methods provided are carried out under shearstress. In various aspects, the shear stress comprises a shear rate ofabout 100 s-1, about 200 s-1, about 300 s-1, about 400 s-1, about 500s-1, about 600 s-1, about 700 s-1, about 800 s-1, about 900 s-1, about1000 s-1, about 2000 s-1, about 3000 s-1, about 4000 s-1, about 5000s-1, about 6000 s-1, about 7000 s-1, about 8000 s-1, about 9000 s-1,about 10,000 s-1, about 11,000 s-1, about 12,000 s-1, about 13,000 s-1,about 14,000 s-1, about 15,000 s-1, about 16,000 s-1, about 17,000 s-1,about 18,000 s-1, about 19,000 s-1, and about 20,000 s-1. In certainaspects, the shear stress comprises a shear rate of about 100 to about10,000 s-1. In other aspects, the shear stress comprises a shear rate ofabout 1,000 s-1 to about 8,000 s-1. In one aspect, the shear rate isabout 6,000 s-1.

In the methods provided, the rVWF or rADAMTS13 is administered to themammal at any dose, including a variety of doses. The dosage may bebased on body weight, activity of the VWF, activity of the ADAMTS13protease, route of administration, health or condition of the mammalianrecipient, and various factors as known to one of skill in the art.

EXAMPLES

Additional aspects and details of the invention will be apparent fromthe following examples, which are intended to be illustrative ratherthan limiting. Example 1 demonstrates that the susceptibility of humanrVWF to ADAMTS13 cleavage varies among species. Example 2 shows thequantification and detection of ADAMTS13-derived cleavage products inplasma. Example 3 describes the detection of VWF cleavage fragments inplasma from subjects treated with human recombinant VWF. Example 4describes the detection of VWF cleavage fragments afterADAMTS13-mediated VWF proteolysis under shear stress. Example 5describes the detection of the effect of recombinant ADAMTS13 onendogenous VWF in plasma.

Example 1 Susceptibility of Human Recombinant Von Willebrand Factor toADAMTS13 Cleavage Varies Among Species

As set out herein above, VWF multimeric size and consequently VWFactivity is regulated by ADAMTS13 activity in the blood. The aim of thestudy was to determine the susceptibility of human rVWF to cleavage byADAMTS13 present in the plasma of different animal species. The abilityof ADAMTS13 of different species to cleave human rVWF was tested invitro as well as in vivo.

Determination of ADAMTS13 Activity: Diluted plasma samples from variousanimals were treated with barium chloride to activate ADAMTS13. HumanrVWF (1 U/mL) (obtained) from CHO cells by fermentation and purification(Turecek et al., Blood 108: Abstract 1017, 2006; American Society ofHematology Annual Meeting Abstracts) was added and the mixture wasincubated for 24 h under denaturing conditions (1.5 M urea). ADAMTS13activity was then determined by a number of assays as described hereinbelow.

Collagen Binding Activity (VWF:CBA) Assay: Samples were incubated inwells coated with collagen. Bound rVWF was detected using a polyclonalantihuman VWF antibody (DAKO, Denmark). The ADAMTS13 activity of plasmasamples was expressed as a percent of residual VWF:CB activity comparedto non-cleaved rVWF and data were illustrated in dose-response curves(1:20 to 1:3000 dilutions) or as a percent of collagen binding activity(CBA) decrease compared to normal human plasma (NHP) (1:20 dilution,equivalent to 50 mU/mL human ADAMTS13 activity). As described hereinabove, the VWF:CBA is a functional assay which provides quantitative aswell as qualitative information on the quality of VWF present.

Fluorescence Resonance Energy Transfer (FRET) Activity Assay: Theproteolytic activity of ADAMTS13 was measured using afluorescent-labeled synthetic VWF peptide composed of 73 amino acids(FRETS-VWF73), Peptide Institute, Inc., Osaka, Japan) according to themanufacturer's instructions. Plasma samples (ADAMTS13 activity) weremeasured against a reference curve prepared from diluted normal humanplasma (NHP) and expressed as a percent of NHP.

Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) andImmunoblottinq: SDS-PAGE was performed under reducing and non-reducingconditions using gradient (3%-8%) Tris-acetate gels followed byelectro-blotting proteins onto PVDF membranes and incubating withmonoclonal (MoAb. VW33-5: TaKaRa Bio Inc., Japan; MoAb N10: Abcam, USA,Kato et al. (2006) Transfusion, 46, 1444) and polyclonal rabbitanti-human VWF (DAKO, Denmark) antibodies. As a control, rVWF wastreated with recombinant ADAMTS13 (Plaimauer et al., Blood 100: 3626,2002). Secondary antibodies were labeled either with alkalinephosphatase (ALP) or horseradish peroxidase (HRP). The proteins werevisualized using an ALP or enhanced chemiluminescence (ECL) detectionkit. Multimer analysis was performed using high resolution horizontalSDS-agarose gel electrophoresis followed by immunostaining with apolyclonal rabbit anti-human VWF antibody.

Animals: Animal plasma samples used in this study were taken from monkey(Rhesus and Cynomolgus), rabbit (New Zealand White), pig (Yorkshire),dog (Beagle), guinea pig (Dunkin Hartley), rat (Sprague Dawley), andmice (VWF-deficient (def) and ADAMTS13-deficient (both with C57BLbackgrounds).

In vivo studies: New Zealand White rabbits were treated with 1200 IUVWF:Ristocetin Cofactor activity (VWF:RCo)/kg body weight (BW) viaintravenous injection. As described herein above, the VWF:RCo assay isone method of measuring VWF concentration/activity. Consequently, theconcentration of VWF is often reported in VWF:RCo units. Blood samplesbefore injection with rVWF and at various time points after injectionwere withdrawn from rabbits from the central auricular artery. Citratedplasma was prepared and stored frozen. Mice were either treated with2000 IU VWF:RCo/kg BW alone or co-treated with rADAMTS13 at 19.4 μg/kgBW via intravenous injection. Blood samples from mice were withdrawn bycardiac puncture and citrated plasma was prepared and stored frozen.Mouse plasma samples from the in vivo studies were immuno-depleted withprotein G sepharose beads before loading gels to avoid reactivity ofMoAbs with endogenous IgGs.

Cleavage of rVWF by Human ADAMTS13

The changes in multimeric structure of rVWF after cleavage by humanADAMTS13 are shown in FIG. 1. VWF multimers in normal human plasma (NHP)show satellite bands, as a result of ADAMTS13 cleavage (see FIG. 1A).Recombinant VWF contains high MW multimers without any satellitestructure (see FIG. 1B). Incubation of rVWF with ADAMTS13 underdenaturing conditions leads to the reduction of multimer numbers withappearance of lower multimers and satellite bands.

The specific cleavage of rVWF monomers by ADAMTS13 is shown in FIG. 2.ADAMTS13-specific cleavage of the rVWF monomers results in a 140 kDaN-terminal and a 176-kDa C-terminal fragment, which can be detected byimmunoblotting. Results of immunoblotting using reducing SDS-PAGE withmonoclonal antibodies for VWF are shown in FIG. 2A. Monoclonal antibodyN10 detects the N-terminal 140-kDa fragment of VWF only when cleavedbetween Tyr¹⁶⁰⁵ and Met¹⁶⁰⁶ by ADAMTS13 (no reaction with the intactVWF). Monoclonal antibody VW33-5 detects the 176-kDa C-terminal fragmentand the intact VWF. Results of immunoblotting using non-reducingSDS-PAGE with a polyclonal antibody for VWF are shown in FIG. 2B. Thepolyclonal rabbit anti-VWF Ab detects both cleavage fragments and theintact VWF multimers.

In Vitro Cleavage of rVWF by ADAMTS13 of Different Animal Species

ADAMTS13-dependent cleavage of rVWF was detected by residual VWFcollagen-binding (VWF:CBA) activity (see FIG. 3). ADAMTS13 activity, asmeasured by FRETS and VWF:CBA, is also set out in FIG. 4. ADAMTS13activity is expressed as a percentage of NHP. The enzymatic activity ofADAMTS13 in rabbit plasma was as high as that of human plasma (asmeasured by CBA and FRETS). Less ADAMTS13 activity was observed insamples from Cynomolgus and Rhesus monkeys, pig, and dog. However, FRETSshowed higher ADAMTS13 activity than CBA. Little or no ADAMTS13 activitywas detected in plasma samples derived from VWF-deficient mouse,ADAMTS13 deficient mouse, rat, and guinea pig.

Visualization of VWF cleavage by ADAMTS13 using high resolution multimeranalysis (see FIG. 5) showed that rabbit plasma induced degradation ofrVWF and the formation of satellite bands similar to normal human plasma(NHP). Cynomolgus monkey, Rhesus monkey, pig, and dog also showedspecific cleavage of rVWF, however, to a lesser extent. No relevantchanges were observed with guinea pig, rat, and mouse plasma comparedwith rVWF incubated with buffer.

The use of immunoblotting to visualize ADAMTS13 cleavage of rVWF (seeFIGS. 6 and 7) demonstrated that the intensity of the bands (cleavedVWF) generally correlated well with the results of the VWF:CBA assay.FIG. 6 shows the results of SDS-PAGE under non-reducing conditionsfollowed by immunoblotting with polyclonal anti-human VWF antibodylinked with HRP. This polyclonal anti-human VWF antibody detects bothVWF cleavage fragments (140 and 176 kDa) and the intact multimers, andis more sensitive but less specific than the monoclonal antibodies. Theresults of immunoblotting with the polyclonal antibody (see FIG. 6)indicated cleavage of human rVWF after incubation with plasma fromhuman, rabbit, monkey, pig, or dog, but not with guinea pig, rat, ormouse.

A comparable result was obtained with the monoclonal antibody VW33-5(see FIG. 7). FIG. 7 shows the results of SDS-PAGE under reducingconditions followed by immunoblotting with monoclonal VWF antibodies. Asdiscussed herein above, monoclonal antibody VW33-5 detects theC-terminal 176 kDa fragment and the intact VWF, whereas monoclonalantibody N10 detects the N-terminal 140 kDa fragment of VWF, only whencleaved between Tyr¹⁶⁰⁵ and Met¹⁶⁰⁶ by ADAMTS13. ADAMTS13 cleavage ofVWF was also detected by the N10 antibody (see 140 kDa bands in FIG. 7).The asterisk in FIG. 7 denotes reactions of the goat anti-mouse IgGantibody (secondary) with endogenous mouse plasma IgGs. These reactionsprecluded visualization of the 140 kDa monomer with the N10 antibody inthe mouse samples. The intensity of the bands generally correlated wellwith results from CBA assays.

In plasma of human, rabbit, Cynomolgus and Rhesus monkeys,ADAMTS13-specific cleavage of the rVWF monomers was demonstrated byimmunoblotting. In pig and dog, low levels of the 176 kDa fragment weredetectable. No rVWF fragments were visible when incubated with plasma ofC57BL mouse strains, rat, and guinea pig.

In Vivo Cleavage of rVWF by ADAMTS13 of Different Animal Species

Specific in vivo cleavage of rVWF (1200 I U VWF:RCo/kg) by ADAMTS13 (140kDa and 176 kDa fragments) in rabbits was detected with monoclonalantibodies by immunoblotting after reducing SDS-PAGE (see FIGS. 8A andB). As controls, uncleaved rVWF and rVWF cleaved with rADAMTS13 in vitroare shown. Characteristic changes in multimer pattern shortly after rVWFadministration are also seen (see FIG. 8C).

FIG. 9 shows specific in vivo cleavage of rVWF (2000 IU VWF:RCo/kg) byADAMTS13 in VWF-deficient and ADAMTS13-deficient mice. The 176 kDa VWFcleavage fragment was not visible in either mouse strain using themonoclonal antibody specific to the C-terminal fragment (VW33-5) (FIG.9A) after non-reducing SDS-PAGE. The asterisk denotes reactions of thegoat anti-mouse IgG antibody with mouse plasma IgGs. These reactionsprecluded visualization of the 140 kDa monomer with the N10 antibody. Nodetectable changes in rVWF multimer pattern were observed (FIG. 9B). 140kDa and 176 kDa homodimers were only detectable in VWF-deficient, butnot in ADAMTS13-deficient mice using the more sensitive (but lessspecific) polyclonal antibody under non-reducing conditions (FIG. 9C).

A direct comparison of the efficiency of ADAMTS13 cleavage in the animalmodels was carried out under non-reducing SDS-PAGE with equal amounts ofVWF:Ag being loaded (see FIG. 10). In rabbit plasma samples, a strongerband corresponding to the 176 kDa VWF cleavage product was detectablecompared to the VWF-deficient mouse sample. No cleavage was detectablein ADAMTS13-deficient mouse plasma. Co-injection of rVWF with humanrADAMTS13 induced cleavage of rVWF. Control lanes 1-3 in FIG. 10represent rVWF uncleaved (1), rVWF premixed with rADAMTS13 (2), and rVWFcleaved in vitro by rADAMTS13 (3).

VWF cleavage fragments were detected in Cynomolgus plasma after a singledose injection of rVWF (100 IU/kg) (see FIG. 11). Completely andpartially ADAMTS13-degraded rVWF fragments (1 Ag U/mL) were measured inCynomolgus plasma after a single dose injection of rVWF with Westernblotting under non-reducing conditions after 15 seconds of exposure withthe rabbit anti-human VWF antibody (FIG. 11A). A baseline degradationband (176 kDa dimer) was seen in Cynomolgus plasma before rVWFinjection. An increase in the amount of the 176 kDa VWF dimer was seenafter the injection of rVWF. The 176 kDa VWF dimer was greater thanbaseline (pre-injection) even 24 h after VWF injection (FIG. 13A), whenno elevated VWF antigen (VWF:Ag) was measured (FIG. 11B).

These results show that substantial differences in the in vitro cleavagesusceptibility of human rVWF by ADAMTS13 were found among the plasma ofdifferent animal species. Rabbit plasma was as effective in proteolysisof human rVWF as homologous human plasma. Plasma samples from Cynomolgusmonkey, Rhesus monkey, pig, and dog showed medium ADAMTS13 proteolyticactivity toward human rVWF. Plasma samples from VWF-deficient mouse,rat, and guinea pig showed virtually no ADAMTS13 activity towards humanrVWF.

The in vivo studies with injection of high doses of rVWF in rabbits andmice confirmed the in vitro results. The residual cleavage observed inthe VWF-deficient mouse was confirmed by the absence of cleavage in theADAMTS13-deficient mouse. In rabbit plasma, rVWF was efficiently cleavedas demonstrated by ADAMTS13-specific cleavage fragments of VWF andsatellite band formation of multimers. In VWF-deficient andADAMTS13-deficient mouse plasma, hardly any rVWF cleavage was noted.Human rADAMTS13 was able to substitute for the mouse ADAMTS13 enzyme andcleave VWF when co-injected with rVWF in mice.

These data demonstrate poor species compatibility between human rVWF andendogenous ADAMTS13 present in plasma of mice, rats, and guinea pigs.ADAMTS13 present in the plasma of the Cynomolgus monkey, Rhesus monkey,pig, and dog showed better proteolytic activity toward human rVWF;however ADAMTS13 proteolytic activity was still lower than that of humanADAMTS13 activity toward human rVWF. Rabbit ADAMTS13 activity appearedto be as effective in proteolysis of human rVWF as homologous humanplasma. These species differences in the proteolyticactivity/specificity of ADAMTS13 should be taken into consideration whenevaluating the efficacy, (patho)physiology, and metabolism of human rVWFin different animals. This study also suggests that the rabbit, andrabbit plasma in general, may be useful as a model for evaluating andtesting human rVWF.

The method is also suitable for detecting the effect of endogenousADAMTS13 on injected rVWF in various animal models. The methods of theinvention allow for the investigation of the suitability of variousanimal models in determining the effect of ADAMTS13 on rVWF in differentspecies.

Example 2 Quantification and Detection of ADAMTS13-derived CleavageProducts in Plasma

The aim of the study was to develop an assay to determine ifADAMTS13-mediated cleavage of VWF (i.e., VWF fragments) could bedetected in plasma samples of Type III VWD subjects after treatment withrVWF during a clinical study.

Normal human plasma, severe VWF-deficient human plasma,ADAMTS13-deficient human plasma, and Cynomolgus plasma samples, obtainedbefore and after (1, 3, 9, 15, and 24 hours) rVWF injection (100 IUVWF:RCo/kg), were used. Completely and partially degraded rVWF treatedwith rADAMTS13 under denaturing conditions were used as controls.

Plasma samples were applied to SDS-PAGE under non-reducing conditionsfollowed by Western blotting. The blots were stained with an HRP-labeledrabbit anti-human VWF polyclonal antibody (Dako), and developed with anenhanced chemiluminescence (ECL) plus technique.

Completely and partially ADAMTS13-degraded rVWF (1 Ag U/mL) weremeasured in buffer and VWF-deficient plasma with Western blotting undernon-reducing conditions after 15 seconds of exposure with the polyclonalrabbit anti-human VWF antibody (see FIG. 12). Reducing conditions werenot sensitive enough to visualize results. No differences betweendilutions in buffer (FIG. 12A) and VWF-deficient plasma (FIG. 12B) weredetected with Western blot analysis. The VWF-derived C-terminal fragment(176 kDa dimer) was detected using ECL. Completely degraded rVWF (1 AgU/mL) could be detected at >62.5 fold dilution (16 nL/μL plasma). Nocross reaction was seen with other human plasma proteins.

VWF cleavage fragments were detected in plasma by increasing thesensitivity of the assay through increasing the exposure time (see FIG.13). Completely and partially ADAMTS13-degraded rVWF (1 Ag U/mL) weremeasured in buffer and VWF-deficient plasma with Western blotting undernon-reducing conditions after 15 seconds (FIG. 13A) and 60 seconds (FIG.13B) of exposure with the rabbit anti-human VWF antibody. A 176 kDacleavage fragment can be seen in normal human plasma, but not inVWF-deficient plasma (see FIGS. 13A and B). Traces of a 176 kDa cleavagefragment are detected in ADAMTS13-deficient plasma (see FIGS. 13A andB), but in a much lesser amount compared to the total VWF present.Completely degraded rVWF (1 Ag U/mL) was well detected at up to 320times dilution (2.5 nL/0.8 μL plasma/lane) with 60 sec exposure time(see FIG. 13B).

The detection limit or level of sensitivity of the Western blot assaywas further determined by diluting plasma samples and increasingexposure time to two minutes (FIG. 14). Normal human plasma was diluted20-fold (from 5 nL to 100 nL) and a 176 kDa cleavage product was welldetected after two minutes when 20-40 nL normal human plasma was applied(in 0.8 μL VWF-deficient plasma) (0.025-0.05 Ag U/mL VWF) (see FIG.14A). No ADAMTS13-specific band (VWF cleavage product) could be seen inVWF-deficient plasma (FIG. 14A). Fully degraded rVWF (1 Ag U/mL) wasdiluted 40-fold (0.5 nL to 20 nL) and could be detected, as measured bythe 176 kDa VWF dimer, at about 0.0006 U/mL concentration (0.5 nL/0.8 μLplasma) (see FIG. 14B).

VWF cleavage by ADAMTS13 was further measured in plasma by quantifyingVWF degradation bands (FIG. 15). A 176 kDa VWF cleavage product was welldetected in normal human plasma at 20 nL applied in 0.8 μL ofVWF-deficient plasma (0.025 Ag U/mL VWF) (see FIG. 15A). Approximately1-2% of C-terminal dimers (the 176 kDa ADAMTS13 specific cleavageproduct) were found in human normal plasma when calculated from areference curve constructed from the band intensity of the differentamounts of completely degraded rVWF (1 Ag U/mL) (see FIG. 15B).

Therefore, a highly sensitive method for the specific detection ofADAMTS13-mediated VWF cleavage bands in plasma was developed. Based on aquantitative comparison with a completely degraded rVWF, approximately1-2% of total VWF:Ag appeared as a 176 kDa degradation band in normalhuman plasma. C-terminal dimers (176 kDa dimers) in normal plasma can bedetected at a lower limit of 0.025-0.05 U/mL VWF:Ag concentration.

This study showed that the VWF cleavage product correlates with ADAMTS13activity indicating that this method is suitable for detecting theeffect of endogenous ADAMTS13 on injected rVWF in vivo. The method canalso be used as a marker of in vivo ADAMTS13 activity. This exampletherefore illustrates the development of a highly sensitive method formeasuring ADAMTS13 activity in vivo by the examination of VWF cleavageproducts in plasma samples.

Example 3 Detection of VWF Cleavage Fragments in Plasma from SubjectsBefore and After Treatment with RVWF

In the course of a clinical phase I trial, type III VWD subjects weretreated with 7.5 IU VWF:RCo/kg body weight of rVWF. Blood samples werecollected before the treatment and at various time points after thetreatment up until 96 h and assayed for the presence ofADAMTS13-dependent VWF cleavage fragments.

The respective plasma samples were applied to SDS-PAGE undernon-reducing conditions on 3-8% Tris-Acetate gels (500 nL per lane)followed by Western blotting using an HRP-labeled rabbit anti-human VWFpolyclonal antibody (Dako) in combination with an ECL plus technique.VWD plasma (VWD-P, George King), normal human plasma (0.1 U/ml dilutedin VWD plasma, NHP), rVWF (0.1 U/ml diluted in VWD plasma), and in vitrodigested rVWF (1 nL diluted in VWD plasma, degradation control) servedas controls. The 176 kDa dimer was not detectable in the pre-treatmentsample, but was clearly discernible in the post-treatment samples of the15 min, 30 min, and 1 h time points (see FIG. 16A). As this fragment wasabsent from the rVWF, the subject's endogenous ADAMTS13 must havecleaved the administered rVWF.

In the course of a clinical phase I trial, type III VWD subjects weretreated with 20 IU VWF:RCo/kg body weight of rVWF (a greater dose thanset out above). Blood samples were collected before the treatment and atvarious time points after the treatment up until 96 h and assayed forthe presence of ADAMTS13-dependent VWF cleavage fragments.

The respective plasma samples were applied to SDS-PAGE undernon-reducing conditions as described above. The 176 kDa dimer was notdetectable in the pre-treatment sample, but was clearly discernible inthe post-treatment samples up to the 32 h time point (see FIG. 16B). TheADAMTS13-specific cleavage fragment was discernible in plasma by Westernblotting for a longer time because of the greater dose of rVWF (20 vs.7.5 IU/kg) administered.

Example 4 Detection of VWF Cleavage Fragments after ADAMTS13-mediatedVWF Proteolysis under Shear Stress

Shear-induced proteolytic cleavage of VWF was performed in a cone-plateviscometer (HAAKE Rheo Stress 1, Thermo Fisher Scientific, Waltham,Mass., USA) in a total volume of 500 μL using a 60 mm cone (0.5° angle).The experimental design was based on a publication by Shim et al. (Blood111:651-657, 2008). rVWF (1 IU/mL final concentration) was mixed withrADAMTS13, plasma-derived ADAMTS13 (pADAMTS13), or normal human plasma(0.2 U/mL final concentration) in a reaction buffer containing 50 mMHEPES, 150 mM NaCl, 0.1 μM ZnCl₂, and 5 mM CaCl₂, pH=7.4. The reactionwas started by subjecting the samples to a shear rate of 6000 s⁻¹ at 37°C. for 15 minutes and was stopped by the addition of EDTA (5 mM finalconcentration). As a negative control, identical samples were similarlyincubated in the absence of shear stress. As a positive control, rVWFwas incubated with rADAMTS13 under denaturing conditions (1.5 M urea)for 24 hours.

The samples were applied to SDS-PAGE under non-reducing conditions on3-8% Tris-Acetate gels (500 nL per lane) followed by Western blottingusing an HRP-labeled rabbit anti-human VWF polyclonal antibody (Dako) incombination with an ECL plus technique. Results of this experiment areshown in FIG. 17. The degradation control marks the position of the 176kDa dimer, which is specifically generated upon ADAMTS13 cleavage ofVWF. Bands of the same mobility were discernible for the samplessubjected to shear stress. By contrast, rVWF starting material and theotherwise identical samples kept under static conditions did not showthe specific cleavage fragment.

The specificity of the observed VWF cleavage by ADAMTS13 was alsoanalyzed by multimer analysis. rVWF (3 IU/mL final concentration) wasmixed with rADAMTS13 (5 U/mL final concentration) in a reaction buffercontaining 50 mM HEPES, 150 mM NaCl, 0.1 μM ZnCl₂, and 5 mM CaCl₂,pH=7.4. The reaction was started by subjecting the sample to a shearrate of 6000 s⁻¹ at 37° C. for 30 minutes and was stopped by theaddition of EDTA (5 mM final concentration). As a negative control, anidentical sample was similarly incubated in the absence of shear stress.

Low and high resolution multimer analysis (see FIGS. 18A and 18B) showedthat rADAMTS13 induced degradation of rVWF and the formation ofsatellite bands, resulting in a pattern similar to that of normal humanplasma (NHP, George King). No relevant changes in the multimer patternwere observed for the sample kept under static, yet otherwise identical,conditions.

The combined data demonstrate that ADAMTS13-dependent VWF cleavage,subject to in vitro conditions that more closely resemble thephysiological one in blood circulation, is achieved and can be measuredby the newly developed assay.

This assay is therefore also suitable for measuring ADAMTS13 activity inhuman plasma samples by the addition of rVWF as a substrate. Applicableare plasma samples with a wide range of ADAMTS13 activity levels.

Example 5 Detection Of The Effect Of Recombinant ADAMTS13 on EndogenousVWF in Plasma

The aim of this study is to further develop the method of detectingADAMTS13 cleavage products in plasma in order to detect the effect ofinjected recombinant ADAMTS13 (rADAMTS13) on endogenous VWF in human anddifferent animal samples. This will result in a method to choose thebest suitable model for preclinical studies of rADAMTS13. Methods as setout herein above are used to test the effect of rADAMTS13 on thecleavage of rVWF and endogenous VWF in the plasma of various animalspecies, including human plasma.

The invention has been described in terms of particular embodimentsfound or proposed to comprise specific modes for the practice of theinvention. Various modifications and variations of the describedinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the relevant fields are intended to be within the scope ofthe following claims.

What is claimed is:
 1. A method for testing effectiveness of a vonWillebrand Factor (VWF) used in treating von Willebrand disease (VWD) ina subject comprising measuring VWF cleavage fragments in a blood samplefrom the subject before and after treatment, wherein an increase in VWFcleavage fragments after the treatment indicates that endogenousdisintegrin and metalloproteinase with a thrombospondin type 1 motif,member 13 (ADAMTS13 ) in the subject is cleaving the VWF and wherein adecrease or absence of VWF cleavage fragments after the treatmentindicates that endogenous ADAMTS13 in the subject is not cleaving theVWF;wherein a change in VWF cleavage fragment level is detected bymeasuring the level of one or more of a 140 kDa VWF fragment or a 176kDa VWF fragment; and the measuring of VWF cleavage fragment levelcomprises performing Western blot analysis with a VWF antibody tovisualize VWF cleavage fragments via enhanced chemiluminescence (ECL);wherein a change in VWF cleavage fragment level is detected by measuringthe level of one or more of a 140 kDa VWF fragment or a 176 kDa VWFfragment; and the measuring of VWF cleavage fragment level comprisesperforming Western blot analysis with a VWF antibody to visualize VWFcleavage fragments via enhanced chemiluminescence (ECL).
 2. The methodof claim 1, wherein the Western blot analysis is carried out undernon-reducing conditions.
 3. The method of claim 1, wherein VWF fragmentsare visualized through use of a VWF antibody conjugated to a marker. 4.The method of claim 3, wherein the marker is alkaline phosphatase (ALP)or horseradish peroxidase (HRP).
 5. The method of claim 1 wherein theVWF antibody is monoclonal or polyclonal.
 6. The method of claim 1,wherein VWF cleavage fragments are detected at a sensitivity level ofabout 0.025 to about 0.05 Ag U/mL VWF.
 7. The method of claim 1, whereinthe blood sample is plasma or serum.
 8. The method of claim 7, whereinthe blood sample is plasma.
 9. The method of claim 1, wherein thesubject is a mammal.
 10. The method of claim 9, wherein the mammaliansubject is human, rabbit, monkey, dog, rat, mouse, or pig.
 11. Themethod of claim 10, wherein the subject is human, rabbit, monkey, ordog.
 12. The method of claim 11, wherein the subject is human.
 13. Themethod of claim 1, wherein the VWF fragment is a 176 kDa VWF fragment.14. The method of claim 1, wherein the VWF cleavage fragments aregenerated by the activity of the ADAMTS13 and carried out under shearstress.
 15. The method of claim 14, wherein the shear stress comprises ashear rate of 100 to 10,000 s-1.
 16. The method of claim 15, wherein theshear rate is 1,000 s-1 to 8,000 s-1.
 17. The method of claim 15,wherein the shear rate is 6,000 s-1.
 18. The method of claim 14, whereinthe shear stress comprises a shear rate of 100 to 10,000 s-1 at atemperature of 20 ° C. to 40 ° C. for a period of time.
 19. The methodof claim 18, wherein the temperature is 30 ° C. to 40 ° C.
 20. Themethod of claim 19, wherein the temperature is 37 ° C.
 21. The method ofclaim 18, wherein the period of time ranges from 30 seconds to 1 hour.22. The method of claim 21, wherein the time ranges from 15 minutes to30 minutes.
 23. The method of claim 22, wherein the time is 30 minutesor is 15 minutes.